Pixel arrays

ABSTRACT

The invention relates to the detection of biomolecules or analogs thereof in micro-arrays and the supports used for the micro-arrays. Methods for determining or testing binding of a first member molecule within an array or library of tentative first member binding molecules for binding with a second member binding molecule are disclosed. A support for a micro-array suitable for determining binding of a first member molecule within a library of spots of tentative first member binding molecules with a second member binding molecule is disclosed. The support includes a support surface wherein surface patches are interspersed within surface areas that are materially distinct from the patches.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of PCT/NL02/00097, filed Feb.15, 2002, designating the United States of America, corresponding to PCTInternational Publication WO 02/066984 (published in English on Aug. 29,2002), the contents of which are incorporated herein in its entirety.

TECHNICAL FIELD

[0002] The invention relates to the detection of (bio)molecules, oranalogues thereof, in micro-arrays and the supports used for themicro-arrays. More particularly, the invention relates to methods fordetermining or testing binding of a first member molecule within anarray or a library of tentative first member binding molecules with asecond member binding molecule, wherein the first and second moleculesare each members of a binding pair. The invention further relates toenzyme-linked detection of the binding pair in high-density micro-arraysystems.

BACKGROUND

[0003] Interactions, or the formation of a specific binding pair,between binding molecules, which in general are bio-molecules, and theircorresponding ligands, which in general are also bio-molecules, arecentral to life. Cells often bear or contain receptor molecules thatinteract or bind with a hormone, a peptide, a drug, an antigen, aneffector molecule or with another receptor molecule. Examples of bindingpairs include: enzymes that bind with their respective substrate;antibody molecules that bind with an antigen; nucleic acids that bindwith a protein, and so on. The terms “interact” or “bind” as used hereinare meant to refer to the range of molecular forces between the bindingmolecule and ligand (or the functional parts thereof) as they approacheach other, and may influence each other's properties. This approachtakes the binding molecule and its ligand through various stages ofmolecular recognition comprising increasing degrees of intimacy andmutual effect, thus the two members bind and form a pair.

[0004] Binding molecules have this binding ability because the bindingmolecules have distinct binding sites that allow for the recognition ofthe ligand in question. The ligand, in turn, has a corresponding bindingsite, and when the two binding sites interact—essentiallyspatial—complementarity, the two molecules can bind. Molecules typicallyhave three dimensions and binding sites are often of a three dimensionalnature, wherein one molecule includes one or more surface projections orprotuberances as one binding site that corresponds to one or morepockets or depressions on the other binding molecule, such as athree-dimensional lock-and-key arrangement that may be an induced-fitvariety.

[0005] Due to the central role binding molecules and their ligands playin life, there is an ever expanding interest in testing for oridentifying the nature or characteristics of the binding site and themembers of the binding pair of molecules involved in such a site. Notonly is one interested in the exact nature of the particular interactionbetween the binding molecule and ligand in question, e.g., in order toreplace or supplement binding molecules or ligands when needed, one isalso interested in knowing the approximating characteristics of theinteraction in order to find, or design, analogs, agonists, antagonistsor other compounds that mimic a binding site or ligand involved.

[0006] Versatile and rapid methods to test for or identify binding pairsand its separate members exist. For instance, most, if not all nucleicacid detection techniques, and molecular libraries using these detectiontechniques entail hybridization of an essentially continuous nucleicacid stretch with a complementary nucleic acid strand, such as DNA, RNAor PNA. Proteins and peptides are often detected using antibodies, orderivatives or synthetic variants thereof. Arrays of biologicalmolecules, i.e., micro-arrays, are used in standard techniques in manylaboratories. Such micro-array-based detection generally includes amethod in which a member of a specific binding pair is detected by meansof an optically detectable reaction. Different supports for thelibraries including tentative or possible first members of the bindingpair (such as nucleic acid, peptide, or of any other nature) are used,but can be divided into two types: porous surfaces and non-poroussurfaces.

[0007] Porous surfaces, such as membranes, cellulose and paper, areprobably the oldest support in use. For example, “dot blots” are widelyused today. The synthesis of macromolecules, e.g., nucleic acids orpeptides, has been described on these porous matrices. Paper was used asa relatively thick, continuous porous matrix on which first memberconstructs were synthesized spot wise. Binding pairs were generallyidentified by detection with, directly or via indirectly, enzyme labeledprobes, thus, allowing increased sensitivity over the use of probes thatwere directly labeled with an optically detectable reporter molecule,such as a fluorescent group. A disadvantage of these methods is that thedensity of spots in these matrices is limited. This limitation iscaused, inter alia, by the diffusion of the enzymatically changedsubstrate in the matrix. To avoid the diffusion and, thus, theinaccurate localization of a binding pair in the field of peptidesynthesis, methods using polyethylene pins (Geysen, 1983) or inpolypropylene wells (Slootstra, 1995; 1997) exist. However, these“early” methods have the disadvantage that no high density arrays(approximately up to not more than 10-20 spots/cm2) can be facilitatedfor various reasons.

[0008] The existence of limited spot densities in the arrays is thereason why more nonporous surfaces are becoming more widely used. In thefield of genomics, huge arrays of polynucleotide sequences are spottedon a variety of surfaces, typically glass slides covered with differentcoatings (See, U.S. Pat. Nos. 6,015,880 and 5,700,637). Array densitiesof 1000 spots/cm2 are possible. Even higher densities are possible whenthe biomolecules (i.e., polynucleotide sequences) are synthesizedin-situ (See, U.S. Pat. No. 5,871,928). For example, in a traditionalgene expression assay designed to profile the expression of many genesin parallel, mRNA is prepared from two different tissue types, e.g.,normal and diseased samples. The isolated mRNA represents a snapshot ofthe current state of expression within the cells. The mRNA is convertedto DNA via a first strand cDNA labeling reaction. After target DNA isdeposited onto coated glass slides and directly labeled cDNA probes arehybridized to the arrays, the hybridized arrays are imaged using anarray scanner and the results are examined for differences in expressionlevels using several image and data analysis software tools. A moreintricate porous support surface has been described for these purposes(See, PCT International Publication WO 00/56934) that uses continuousporous matrix arrays. On microscope slides, a continuous slab ofpolyacrylamide is formed, e.g., 20 um thick and having a thin,continuous porous matrix (hydrogel) that is combined with a non poroussurface (glass).

[0009] Detection of specific binding pairs on or in the high-densitysupports is achieved with directly labeled probes that include opticallydetectable, e.g., fluorescent, nucleotides or antibodies. The detectiontechniques are highly sensitive, have low non-specific binding and highphoto stability. Labeled nucleotides are widely used for labeling DNAand RNA probes, especially for multicolor analysis in micro-arrays, butalso for FISH, chromosome identification, whole chromosome painting,karyotyping and gene mapping.

[0010] Labeled nucleotides are available in a range of bright, intensecolors having narrow emission bands that are ideal for multiplexingwithin a single sample. For protein or peptide detection, fewerfluorescently labeled probes are available since the field of protein orpeptide based high-density micro-array systems is not as well developedas micro-arrays based on nucleic acid detection.

SUMMARY OF THE INVENTION

[0011] In one exemplary embodiment, an array or library of first membersis disclosed, in general spatially and/or addressable bound, most oftencovalently, to such a support, e.g., by spotting or gridding. A secondmember, the detecting or specific binding molecule, which is directly orindirectly labeled with a marker molecule (such as a fluorescentcompound) to facilitate optical detection of the aforementioned pair isalso disclosed to detect the putative first members of the array withwhich it can bind. The second member can of course be a nucleic acid, areceptor molecule, an antibody or the like. Binding of the secondmember, thus, identifies the first member because of its specificlocalization on the support.

[0012] The invention combines the advantages of high density arraying,i.e., testing a lot of binding events at once, and enzyme-linked assays(very sensitive), thus, allowing for the detection of more binding pairsmore rapidly. Micro-array systems are disclosed herein that work withenzyme-linked assays in order to detect the molecule of interest on ahigh-density support. Such testing of high densities of constructs on asolid support in a enzyme-linked assay is disclosed by the invention,wherein a first member is attached to or synthesized on one side of asurface of the support in a density of at least 25, at least 50, atleast 100, about 200-500, or even 1000 spots per square centimeter.

[0013] In one exemplary embodiment, the invention describes a support ofpolymeric material, i.e., a polymeric support, having a library of spotsof the tentative first member binding molecules in a density of at least25 spots, at least 50 spots, at least 100 spots, about 200-500 spots, oreven 1000 spots per square centimeter. In another exemplary embodiment,the polymeric material comprises polypropylene having hydrophilicpatches.

[0014] The first binding pair members may be spotted or gridded in apositionally or spatially addressable way such that many differentconstructs or first member molecules on the support can interact with asecond member or binding molecule. The spots can overlap, as long as theconstituting collection of first member molecules are spatiallyaddressable and distinct. Spotting can be done using piezodrop-on-demand technology or by using miniature solenoid valves.Gridding can be done using a set of individual needles that pick upsub-microliter amounts of segment solution from a microtiter plate,wherein the segment solution includes solutions having the firstmembers.

[0015] When peptides are tested, the support may be de-protected andextensively washed after the linking reaction to remove uncoupledpeptides. The disclosed method gives a peptide construct density aslarge as 25 to 50, even 100 to 200, or up to 500 to 1000 spots persquare centimeter. This density allows for the screening of a largenumber of possible peptide constructs of the proteins that bind with anantibody. For example, in one exemplary embodiment, 25,000 to 100,000constructs are made on a 1000 square centimeter surface. The surface isscreened for binding in an enzyme-linked assay, be it directly orindirectly, wherein a fluorescent substrate is generated with 100 ml ofenzyme-labeled probe solution that contains 1-10 μg of probe/ml. Thescreened surface is developed with an optically detectable substrateusing established techniques.

[0016] The invention, thus, discloses a method for determining thebinding of a first member molecule from a library of tentative firstmember binding molecules with a second member binding molecule. Themethod comprises providing a polymeric (such as the polypropylene typesurface having hydrophilic patches) or plastic support with a library ofspots of the tentative first member binding molecules in a density of atleast 25 spots per square centimeter. The method further comprisesdetecting the binding of the first member molecule with the secondmember molecule with an enzyme-linked assay, wherein theenzyme-linked-assay comprises the production of a fluorescent orchemiluminescent substrate. Fluorescent substrates can be produced witha host of enzyme systems, such as horse-radish-peroxidase, alkalinephosphatase or other known substrate-enzyme systems such as thosedisclosed in Mendoza et al. including “High-throughput microarray-basedenzyme-linked immunosorbent assay (ELISA),” Biotechniques, EatonPublishing, Natick, US vol. 27, (1999), where an optically flat,glass-based support is described, including multitudes of identicallypatterned arrays of antigens printed to the glass.

[0017] As described herein, indirect or direct fluorescence detectionallocates antibody binding constructs. For instance, direct fluorescencedetection with confocal scanning detection methods allows antibodydetection on spots generated with droplets having peptide-solution inthe sub-nanoliter range, which makes even higher construct densitiesfeasible. Nucleic acid libraries may be made in a similar fashion usingenzyme-labeled nucleic acid probes.

[0018] Furthermore, the invention describes a support for a micro-arrayconfigured for testing binding of a first member molecule within anarray or library of tentative first member binding molecules with asecond member binding molecule. The support includes a surface havingpatches that are interspersed within areas, wherein the areas arematerially distinct from the patches. In general, the surface isobtained using various complicated methods that comprise masking andsubsequent photographic exposure and development (See, PCT InternationalPublication WO 94/27719), plasma treatment, polymerization,photo-oxidation or electron beam treatment (See, WO 99/58245). Othertechniques (See, Canadian Publication CA 2260807) require an inert solidsupport material to which the hydrophobic and hydrophilic areas need beapplied, such as by way of coating. Other techniques (See, Great BritainPublication GB 2332273) provide an extremely hydrophobic surface inrelation to the sample solution that is applied after which samples arethought to adhere to the surface by drying. In U.S. Pat. No. 5,369,102,a support with two opposing surfaces, one hydrophobic and the opposingone hydrophilic, are provided for the attachment of cells to thehydrophilic surface. PCT International Publication WO 99/32705 disclosesvarious grafting protocols, but does not disclose requirements as toroughness of the surface, or to a pattern of hydrophilic and hydrophobicpatches.

[0019] The present invention has recognized that masking or coating isnot required and that grafting surfaces, such as polypropylene, issuitable provided that at least one side of the surface of the startingmaterial, such as a substantially flat surface of at least 0.5 squarecentimeters or at least 1 square centimeter, is configured with asubstantial roughness characterized by elevations and depressions. Therough surface allows for the interspersed character of hydrophobic andhydrophilic patches to occur on the side or surface. The pattern ofhydrophilic (hydrophilic matrices typically cause severe diffusion) andhydrophobic areas (which may block diffusion) as disclosed hereindiminish diffusion, especially when the patches are smaller than thedroplet size of dispensed material (spots), which are the smallest whenthe spot density is the highest.

[0020] In one exemplary embodiment, the invention discloses a support,wherein the surface of the areas essentially comprises relativelyhydrophobic polypropylene and the surface of the patches essentiallycomprise polypropylene having a relatively hydrophilic material, such agrafted polyacrylic acid. The support disclosed herein comprises atleast a spot or dot, e.g. a collection of first member molecules such asa nucleic acid or peptide construct, density as large as 25, or up to50, 100, 200, 500 or even 1,000 spots per square centimeter. Further,the spots or dots are positionally or spatially addressable, whereineach of the spot or dot covers at least one patch, but the spot or dotmay cover from 3-5, or even from 5-15 or more hydrophilic patches orpixels.

[0021] Although the surface, such as the polypropylene, may not becompletely covered with a homogenous graft, the high loadings of peptideor nucleotide per square centimeter are possible due to the relativelyhigh surface occupation of the grafts, such as polyacrylic acid, on thesurface. Thicker grafts can carry higher peptide or nucleic acidloadings, but may suffer from more diffusion problems of dispensedmaterial because of the growing occupation of grafted surface. Thus, thematerial can be made to suit various needs as regard to loading versusdiffusion.

[0022] The invention further discloses a solid support having at leastone peptide, or at least one nucleotide. The solid support may include aplurality of peptides (or likewise of nucleotides), wherein the peptidesor nucleotides are arranged in spots.

[0023] In another exemplary embodiment, the invention describes a methodfor determining binding of a first member molecule within a library oftentative first member binding molecules with a second member bindingmolecule. The method includes providing at least one surface of asupport having a library of spots of the tentative first member bindingmolecules, detecting the binding in an enzyme-linked assay and providingfor limited, minimalized or restricted diffusion of an opticallydetectable marker molecule. Since diffusion is limited, the enzymaticreaction and the deposit or localization of the resulting (optically)detectable marker molecules can be determined with more precision andallow for higher densities than with previous micro-arrays usingenzyme-linked-detection.

[0024] In a further exemplary embodiment, the invention discloses amethod where the diffusion is limited by providing at least one surfaceof the support having surface patches that are interspersed withinsurface areas, wherein the surface areas are materially distinct fromthe patches. (See, FIG. 1). The invention also provides a support (alsoreferred to as a discontinuous matrix array or pixel array), wherein thesupport surface material is of a varied or discontinuous nature asregards to hydrophilicity. In one embodiment of a support having ahigh-density micro-array, patches of relative hydrophilicity areinterspersed with areas of relative hydrophobicity. There does not needto be a sharp border between the patches and the surrounding area. It issufficient when distinct material differences or discontinuities existbetween the center of a patch and the middle line of a surrounding area,wherein there is a more or less gradual material change in between.

[0025] The patches and surrounding areas may be in a strict matrix orgrid format, but this is not necessary. The patches are at least one ortwo dimensions smaller than the size of the circumference of thepositioned droplets or spots of first member molecules that, in a laterphase, will be provided to the support surface. Since the patches aresmaller than the droplets or spots, at least 3-5 or at least 10-20hydrophilic patches will fit within the circumference of a later spottedspot or droplet of the solution of a first member, whether the firstmember is a nucleic acid, a peptide, any other (bio-)molecule orcombination thereof. The patches resemble pixels that, after a markermolecule has attached to a specific binding pair, create the opticallydetectable image, wherein a spot with a collection of first membermolecules bound to second member molecules is detected.

[0026] In another exemplary embodiment, a one-to-one fit of pixel orpatch to droplet or spot is also feasible even when the patch is largerthan a spot, but this not necessary. It is also not necessary to applyor provide the patches in a regular pattern. When a droplet or spot isprovided, the interspersed hydrophobic character of the support surfacewill limit the diffusion of any aqueous solution. The diffusion of asolution of an optically detectable substrate (be it as precipitate oras solution) formed after the enzymatic reaction has taken place willalso be limited in a later phase. The enzymatic reaction takes placewhen a first member is bound to a second member of a binding pair,wherein the presence of the relatively hydrophilic patch or patcheswithin the droplet or spot circumference allows the substrate to bepositioned, or detectable. The patches disclosed herein may also bedescribed as pixels within the spot(s), wherein the optically detectableor fluorescent substrate will finally be located. If so desired, patchesmay be hydrophobic where the surrounding area is relatively hydrophilic,when, for example, solutions or (optically detectable) markers aretested of a more hydrophobic nature.

[0027] In another exemplary embodiment, the support described hereincomprises at least a spot or dot, e.g., a collection of first membermolecules such as a nucleic acid or peptide construct, density as largeas 25 or 50, even 100, 200, up to 500, or even 1,000 spots per squarecentimeter. The spots or dots are positionally or spatially addressable,wherein each of the spots or dots cover at least one patch, cover from3-5 patches, or even 5-15 or more patches or pixels.

[0028] Hydrophilic path sizes can be modified by selecting theappropriate support material, such as polyethylene, polypropylene oranother relatively hydrophobic plastic material to begin with, or byproviding the patch size with patches in the desired size, such as byutilizing print technology. For instance, a support surface may beproduced from a relatively hydrophobic polypropylene surface upon whichgrafts are provided that form the relatively hydrophilic patches. Thegrafts are made with polyacrylic acid that has an excellent suitablehydrophilic nature and allows for testing under physiologicalcircumstances. The patch size may be influenced by selecting theappropriate roughness or a polyethylene or polypropylene startingmaterial. The roughness can also be modulated by sanding, polishing, anyother mechanical (printing) or chemical (etching) method, orcombinations thereof to modulate a surface on which the hydrophilicpatches are to be generated. The smaller the hydrophilic patch size, thesmaller the droplets can be applied, such as up to the size where atleast one patch falls within the circumference of the applied droplets.

[0029] A method for determining binding of a first member moleculewithin a library of tentative first member binding molecules with asecond member binding molecule is also disclosed. The method includesproviding a support with spots comprising the tentative first memberbinding molecules, providing a second member binding molecule anddetecting binding of a first member molecule with the second memberbinding molecule.

[0030] The binding is detected with an optically detectable marker, suchas a fluorophore, that is directly or indirectly labeled to a probe. Theprobe may be a nucleic acid or an antibody and, thus, allows a supportof the present invention to be used in any type of micro-array. Bypreventing diffusion, problems such as signal overload can be avoided orcircumvented. Thus, one exemplary embodiment of the invention disclosesa method wherein binding pairs are detected via enzyme-linked-assaytechniques where diffusion or leakage can be a problem. By preventingdiffusion, the enzymatic detection method disclosed herein is moresensitive and allows fewer copies of tentative first member molecules tobe spotted on one spot, thus decreasing spot-size and increase spotdensity without losing sensitivity. The enzymatic detection may be up to10-1,000 times more sensitive than detection using directly labeledprobes.

[0031] Suitable enzyme-substrate combinations and methods for use inexemplary embodiments of the invention are, for example, found with U.S.Pat. No. 4,931,223 that discloses processes in which light of differentwavelengths is simultaneously released from two or more enzymaticallydecomposable chemiluminescent 1,2-dioxetane compounds. The compounds areconfigured by means of the inclusion of a different light emittingfluorophore in each compound, such that each compound emits light of thedifferent wavelengths by decomposing each of the compounds with adifferent enzyme. Also, Bronstein et al., BioTechniques 12 #5 (May 1992)pp. 748-753, “Improved Chemiluminescent Western Blotting Procedure”discloses an assay method in which a member of a specific binding pairis detected with an optically detectable reaction. The reaction includesan enzyme of a dioxetane such that the enzyme cleaves anenzyme-cleavable group from the dioxetane to form a negatively chargedsubstituent bonded to the dioxetane. The negatively charged substituentcauses the dioxetane to decompose and form a luminescent substance. Canoet al., J. App. Bacteriology 72 (1992) discloses an example of nucleicacid hybridization with a fluorescent alkaline phosphatase substratethat can also be used in the present invention, and Evangelista et al.,Anal. Biochem. 203 (1992) teaches alkyl- and aryl-substituted salicylphosphates as detection reagents in enzyme amplified fluorescence DNAhybridization assays.

[0032] As will be described herein, a fluorescent substrate for alkalinephosphatase-based detection of protein blots is used with fluorescencescanning equipment, such as Molecular Dynamics FluorImager or Storminstruments, generally known as Vistra ECF and typically deemed suitablefor use in Western blotting, and dot and slot blotting applications. Theenzymatic reaction of alkaline phosphatase dephosphorylates the ECFsubstrate to produce a fluorescent product which is detectable in amethod of the invention. The invention also discloses a method wherein asubstrate for evaluating glycosidic enzymes comprising a fluoresceinderivative, such as disclosed in U.S. Pat. No. 5,208,148, is used. Thesubstrate bears a lypophilic character and will reside in hydrophobicareas of the surface.

[0033] Another exemplary embodiment of the invention discloses asynthetic molecule comprising a binding site, i.e., located on thedetected first member molecule or derivatives thereof, or a bindingmolecule including a binding site identifiable or obtainable by a methodaccording to the invention. In a further exemplary embodiment of theinvention, the support or methods may be used for identifying orobtaining a synthetic molecule having a binding site, or for identifyingor obtaining a binding molecule capable of binding to a binding site,and the use of such an obtained molecule for interfering with oreffecting binding to a binding molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1. Surface structure of polyacrylic acid graftedpolypropylene (PP).

[0035]FIG. 2. ECF-substrate wettability of different surfaces.

[0036]FIG. 3. Storm fluorescence signals of the binding of peptide nr 1,2, 3 and 4 (y-axis) to Mab GO 1 using five different gridding pins (onX-axis diameter gridding pins). Four different peptide concentrationswere spotted on three different grafts: 12/50 Ac, 9/30 Ac and 6/12Acgrafts.

[0037] FIGS. 4A-4D. Maximal fluorescent signals of the spots as detectedby the Storm of the binding of Mab GO1 to the peptide nr 1 (SEQ ID NO:2), 2 (SEQ ID NO: 3), 3 (SEQ ID NO: 4) and 4 (SEQ ID NO: 5) on graft6/12Ac using four different peptide concentrations and five differentgridding pins are shown. Peptide concentrations: FIG. 4A: 1 mg/ml; FIG.4B: 0.2 mg/ml; FIG. 4C: 0.04 mg/ml and FIG. 4D: 0.008 mg/ml.

[0038] FIGS. 5A-5C. Maximal fluorescent signals of the spots as detectedby the Storm of the binding of Mab GO 1 to peptides nr 1 (SEQ ID NO: 2),2 (SEQ ID NO: 3), 3 (SEQ ID NO: 4) and 4 (SEQ ID NO: 5) are shown.(Peptide concentration 0.2 mg/ml) on graft 6/12Ac, 9/30Ac and 12/50Ac.FIG. 5A: graft 6/12Ac; FIG. 5B: graft 9/30Ac; FIG. 5C: graft 12/5OAc.

[0039]FIG. 6A. Schematic presentation of a head-to-tail complete matrixscan. 12345678901 and ABCDEFGHIJK represent sequences derived from aprotein and a schematic presentation of a tail-to-tail complete matrixscan. This scan is similar to the scan shown in FIG. 4, however, thecysteine residue is positioned at the N-terminus of the second buildingblock, leading to a reversed or tail-to-tail orientation of bothbuilding blocks. Both sequences are linked as previously described. Inthis scan, both sequences are shifted independently through the completeprotein sequence, generating a library of all possible sequencecombinations.

[0040]FIG. 6B. List of all peptides (derived from hFSH) containing anN-terminal bromoacetamide group. Peptides 1) through 181) of FIG. 6Bcorrespond to SEQ ID NOS: 6-186, respectively.

[0041]FIG. 6C. List of all peptides (derived from hFSH) containing a C-or N-terminal cysteine. Peptides 1) through 7) correspond to SEQ ID NO:187-193, respectively; peptide 8) corresponds to SEQ ID NO: 12; peptides9) through 16) correspond to SEQ ID NOS: 194-201, respectively; peptide17) corresponds to SEQ ID NO: 22; peptides 18) through 20) correspond toSEQ ID NOS: 202-204, respectively; peptide 21) corresponds to SEQ ID NO:26; peptides 22) through 31) correspond to SEQ ID NOS: 205-214,respectively; peptide 32) corresponds to SEQ ID NO: 36; peptides 33)through 48) correspond to SEQ ID NOS: 215-230, respectively; peptide 49)corresponds to SEQ ID NO: 54; peptides 50) through 59) correspond to SEQID NOS: 231-240, respectively; peptide 60) corresponds to SEQ ID NO: 64;peptides 61) through 70) correspond to SEQ ID NOS: 241-250,respectively; peptide 71) corresponds to SEQ ID NO: 76; peptide 72)corresponds to SEQ ID NO: 251; peptide 73) corresponds to SEQ ID NO: 78;peptides 74) through 85) correspond to SEQ ID NOS: 252-263,respectively; peptide 86) corresponds to SEQ ID NO: 89; peptides 87)through 90) correspond to SEQ ID NOS: 264-267, respectively; peptide 91)corresponds to SEQ ID NO: 95; peptides 92) through 98) correspond to SEQID NOS: 268-274, respectively; peptides 99) and 100) both correspond toSEQ ID NO: 103; peptides 101) and 102) correspond to SEQ ID NOS:275-276, respectively; peptide 103) corresponds to SEQ ID NO: 107;peptides 104) through 133) correspond to SEQ ID NOS: 277-306,respectively; peptides 134) corresponds to SEQ ID NO: 137; peptides 135)and 136) correspond to SEQ ID NOS: 307 and 308, respectively; peptide137) corresponds to SEQ ID NO: 141; peptides 138) through 152)correspond to SEQ ID NOS: 309-323, respectively; peptide 153)corresponds to SEQ ID NO: 157; peptide 154) corresponds to SEQ ID NO:324; peptide 155) corresponds to SEQ ID NO: 159; peptides 156) through164) correspond to SEQ ID NOS: 325-333, respectively; peptide 165)corresponds to SEQ ID NO: 169; peptides 166) through 169) correspond toSEQ ID NOS: 334-337, respectively; peptide 170) corresponds to SEQ IDNO: 173; peptides 171) through 174) corresponds to SEQ ID NOS: 338-341,respectively; peptide 175) corresponds to SEQ ID NO: 179; and peptides176) through 183) correspond to SEQ ID NOS: 342-349, respectively.

[0042]FIG. 6D. Complete matrix scan, i.e., after coupling of all?ALL inB listed in B sequences to all? in C listed in C sequences, exemplifiedby cards 145-155 and a full picture of all binding values of all ca.40,000 peptides (below). Peptide VYETVRVPGCAC$ADSLYTYPVATQ correspondsto SEQ ID NO: 350. The $ symbol refers to a bromoacetamide group.

[0043]FIG. 7A. Schematic presentation of a multi-building block scan.12345678901 (building block 1), NOPQRSTUVWXY (building block 2) andBCDEFGHIJKLM (building block 3) represent successive sequences derivedfrom a protein. Building blocks were linked via a thioether bridge,formed by reaction of a free thiol function of a C-terminal cysteineresidue (C) in building block 1 and a bromoacetamide group ($) at theN-terminus of building block 2 and so on, as described in example 3. Inthis scan all sequences are subsequently shifted simultaneously throughthe complete protein sequence to obtain the complete library.

[0044]FIG. 7B. Working example obtained with an anti-hFSH monoclonalantibody-02.

[0045]FIG. 7C. Binding values and list of peptides coupled onto eachother. Peptides 1 through 36 correspond to SEQ ID NOS: 351-386,respectively. Card 6 is associated with SEQ ID NO: 356, card 07 isassociated with SEQ ID NO: 357, card 08 is associated with SEQ ID NO:358; card 09 is associated with SEQ ID NO: 359; card 0 is associatedwith SEQ ID NO: 360; card 11 is associated with SEQ ID NO: 361; card 12is associated with SEQ ID NO: 362; card 13 is associated with SEQ ID NO:363; card 1 is associated with SEQ ID NO: 371; card 22 is associatedwith SEQ ID NO: 372; card 35 is associated with SEQ ID NO: 385; and card36 is associated with SEQ ID NO: 386.

[0046]FIG. 7D. One square in full detail. The peptide $CKELVYETVRVPG(SEQ ID NO: 1) was coupled to the cysteine of card 06, which isassociated with SEQ ID NO: 356. The $ symbol refers to a bromoacetamidegroup. To this card, peptides 1 to 36 were spotted with gridding pins.The binding values are shown below. Chemistry in short: Polypropylene(PP) surface was gamma irradiated (in this case 50kGy) in the presenceof CuSO₄ and (in this case 12%) acrylic acid. Carboxylic acidfunctionalized PP was treated with Boc-HMDA/DCC/HOBt and subsequenttreatment with trifluoracetic acid (TFA) yielded a surface with aminogroups. To this amino group functionalized PP surface,N-Fmoc-S-trityl-L-cysteine (Fmoc-Cys-(Trt)-OH) was coupled using DCC andHOBt. Subsequently the Fmoc group was removed, followed by acetylationof amino group. Treatment of the surface with TFA (with triethylsilanand water as scavengers) yielded a thiol functionalized surface.Bromoacetyl (or other thiol reactive) containing peptides were allowedto react with the thiol groups of the PP surface in 0.015 M NaHCO₃ (pH7-8, overnight reaction). Subsequently, the StBu groups (of theS-t-butylthio protected Cys residues) of the coupled peptides wereremoved using NaBH₄ (14 mg/ml in 0.015 M NaHCO₃ pH 7-8, 30 min, 30° C.),generating new thiol groups in the peptides. A second set of Bromoacetyl(or other thiol reactive) containing peptides were allowed to couple tothe first set, making peptide constructs. This process can be repeatedseveral times using different sets of bromoacetylated peptides. Peptides1 through 36 corresponds to SEQ ID NOS: 351-386, respectively.

[0047]FIG. 8. Storm fluorescence signals of the binding of Glu-ox to MabGO1 on 3 different grafts using five different gridding pins.

[0048]FIG. 9. On a matrix-scan of human Follicle-Stimulating Hormone(hFSH), the polyclonal anti-hFSH serum R5125 (Biotrent 4560-5215) wastested at 1 ug/ml. The matrix included four large squares (left side ofthe picture). Each large square contains 48 smaller squares. To thethiol group functionalized surfaces of each of these 48 squares (on allthe four plates), one bromoacetylated hFSHpeptide (or a control peptide)is coupled via its bromoacetyl groups as described herein. In this way,each of the overlapping 13-mer peptides covering hFSH are coupled,generating 181 overlapping hFSH peptide functionalized squares+11control peptide squares. All peptides possess a cysteine with a thiolprotecting tert-butylsulfenyl group (Cys(StBu)). The same set ofbromoacetylated hFSH peptides can be coupled to each peptidefunctionalized small square when the protecting StBu groups of thepeptides on the peptide functionalized surfaces is removed by treatmentof NaBH4 in aqueous environment at pH 7-8. Within each peptidefunctionalized square all bromoacetylated hFSH overlapping peptides arespotted generating, after coupling, 181 26-mer hFSH peptide constructs(spots) within each peptide functionalized square. In this way amatrix-scan is generated of all 32,761 (181*181) overlapping FSH 26-merpeptide constructs. The position of the cysteine (Cys(StBu)) in thepeptides, used for coupling, varies. Peptide 1 (first 13-mer ofhFSH=1-12Cys) has a Cys(StBu) on its C-terminal end, peptide 2 (peptideCys2-13 of hFSH) contains a Cys(StBu) on the N-terminal site of thepeptide while peptide 3 (peptide 3-14Cys of hFSH) again has a Cys(StBu)on its C-terminal end. Peptide 4 (peptide Cys4-15 of hFSH) has again anN-terminal Cys(StBu) and so on. Peptide 1 is coupled to the left topsmall square of the left top large square, peptide 2 is coupled to theleft top small square, one step to the right, of the left top largesquare, peptide 3 is coupled to the left top small square, two steps tothe right, of the left top large square and so on. The two enlargedsquares on the right side of the figure show binding of antibody R5125to peptide constructs on peptide functionalized square no.150 (upperenlarged square=peptide 150-162Cys of hFSH) and peptide functionalizedsquare no.66 (lower enlarged square=peptide Cys66-78 of hFSH). A blackcolor represents binding of antibody to peptide (black square) orpeptide constructs (black spots). In the lower enlarged square, thefirst spot (left top) indicates binding of the antibody to a controlpeptide construct, the next spot to the right represents binding to apeptide construct containing peptide no. 1 (hFSH 1-12Cys(StBu)) coupledto hFSH Cys66-78 in lower enlarged square, again one spot to the rightshows binding of the antibody to peptide construct hFSH Cys(StBU)2-13with hFSH Cys66-78 and so on. White spots represent less binding of theantibody to the peptide construct compared to the binding of theantibody to the peptide within the square. No visible spots representequal binding of the antibody to the peptide constructs compared to thebinding of the peptide within the squares.

DETAILED DESCRIPTION

[0049] The micro-array support and methods of the present invention maybe used to detect binding of peptides, nucleic acids or otherbiomolecules. Conventional Pepscan methods use pins (Geysen et al.) orwells (Slootstra et al.). Polyacrylic acid grafts or other acrylicgrafts on the polyethylene pins or in the polypropylene wells were usedas carriers of peptides. Due to the high peptide loadings (each othercarbon atom of the polymer can in theory carry a peptide) tested in anELISA format, extreme low binding-interactions of a peptide to anantibody can be detected (detection of kD<3 M are possible). In thissystem, the interactions were separated physically, i.e., by walls ofwells. Technically, miniaturization of this concept stops atapproximately 10 wells/cm² due to the limitations of conventional(syringe/needle) liquid handling techniques in practice. When the set-upis miniaturized, it is desirable to keep the two strongholds (highpeptide loadings in combination with enzyme-linked detection methods)intact.

[0050] Rough polypropylene (PP) supports are commercially available andare widely used as non-shiny material in all sorts of applications. Therough PP appeared to be an ideal template for attaching polyacrylic acidgrafts. For example, microscope viewing of PP (EVACAST 1070 N16; VinkKunststoffen BV) surfaces reveal rounded elevations (hills) separated bytiny depressions (valleys) (See, FIG. 1). The PP surface on top of thehills is relatively rough compared to the surface of valleys between thehills. The rough surface appeared to be a good scaffold for attachinggrafts whereas the depressions accept grafts less readily. Thus, duringgrafting procedures using gamma irradiation, the graft is not regularlydispersed along the surface, but is deposited in patches surrounded bymaterially different areas corresponding to the depressions in thematerial. For example, using CuSO₄ and acrylic acid during grafting,most of the polyacrylic acid polymers are grafted on the top of theelevations and less in the depressions (See, FIG. 1). Thus, amore-or-less regular pattern of hydrophilic (polyacrylic acid grafts)patches and relatively hydrophobic (places without or less polyacrylicacid grafts) areas are present on the grafted PP surface.

[0051] The pattern of hydrophilic (normally hydrophilic matrixes causesevere diffusion) and hydrophobic areas (blocks diffusion) diminishdiffusion, especially when the patches are smaller then the droplet sizeof dispensed material. Although the surface of the PP is not completelycovered with a homogenous graft, high loadings of peptide/cm² arepossible due to the relatively high surface occupation of thepolyacrylic acid grafts on the PP surfaces. In the above describedsetup, thicker grafts can carry higher peptide loadings, but will sufferfrom more diffusion problems of dispensed material because of thegrowing occupation of grafted surface. However, the material can be madeto suit various needs as regard to loading versus diffusion.

[0052] Enzyme-linked assays use substrates which are converted by theenzyme in products that precipitate in situ or are water soluble. Adrawback of precipitating products is the non-reusability of the systemcaused by insolubility of the precipitated material during cleaning. Theset up that makes use of non precipitating products, in particular nonprecipitating products which are fluorescent, because of the ease ofdetection by modern fluorescent signal detecting applications isdesirable. When substrates (developing soluble products) are put on thesurface, such as where excess of substrate material is in a later stageremoved from the surface, dye development does not suffer from diffusionproblems. This phenomenon is caused by the valley/hill orhydrophobic/hydrophilic construction of the surface in combination withexcellent wettability properties of the polyacrylic acid matrix.

[0053]FIG. 2 shows the Vistra ECF(2′(2-benzthiazoyl}-6′-hydroxy-benzthiozole phosphatebis-(2-amino-2-methyl-1,3-propanediol) salt; Amersham Pharmacia Biotech)substrate wettability of i) with and ii) without poly acrylic acidgrafted PP (EVACAST 1070 N16; Vink Kunststoffen BV) and iii) CMT-glassslides (Corning) as detected on a Storm Fluorimager (MolecularDynamics). Although the polyacrylic acid grafted PP-EVACAST surface isnot continuously occupied with porous (polyacrylic acid grafts)material, the Storm Fluorimager does not detect irregular surfacepatterns. This is in contrast to un-grafted PP-EVACAST or CMT-glassslides.

EXAMPLES Example 1

[0054] A polypropylene (PP) support (EVACAST 1070 N16; Vink KunststoffenBV) was grafted with acrylic acid to introduce polyacrylic acid graftson the PP surface. In this case, the solid PP support was irradiated inthe presence of 6%, 9% or 12% acrylic acid solutions in water containingCuSO4 using gamma radiation at a dose of 12, 30 or 50 kGy (combinations:6% acrylic acid and 12kGy=6/12Ac; 9% acrylic acid with 30 kGy=9/30Ac and12% acrylic acid with 50 kGy=12/50Ac). The grafted solid supportcontaining carboxylic acid groups was functionalized with amino groupsvia coupling of t-butyloxycarbonylhexamethylenediamine (Boc-HMDA) usingdicyclohexylearbodiimide (DCC) with N-hydroxybenztriazole (HOBt) andsubsequent cleavage of the Boc groups using trifluoracetic acid. Tointroduce a thiol reactive bromacetamide group on the support, the aminogroup functionalized support was treated with bromoacetic acid using DCCor DCC/HOBt.

[0055] Peptides containing cysteine residues were able to couple to thebromo functionalized surface via the thiol group of the cysteineresidues forming a stable thioether bond. Peptides were spotted on thebromo functionalized surface using gridding pins (Genomic Solutions)with different diameters (1.5 mm, 0.8 mm, 0.6 mm, 0.4 mm and 0.25 mm).Solutions with different concentrations of peptide were used (1 mg/ml,0.2 mg/ml, 0.04 mg/ml and 0.008 mg/ml). When aliquots of peptidesolutions (in bicarbonate buffer at about pH 7-8) were dispensed on thesupport using the gridding pins, the coupling of the bromo group on thesurface to the thiol group of the peptide was achieved in a humidchamber (overnight reaction). Extensive washing removed uncoupledpeptide.

[0056] Peptides used included: GCASLQGMDTCGK (Nr1) (SEQ ID NO: 2),CAFKQGVDTCGK (Nr2) (SEQ ID NO: 3), APDPFQGVDTCGK (Nr3) (SEQ ID NO: 4),and GCAPDPFQGVDTCGK (Nr4) (SEQ ID NO: 5). From surface plasmon resonance(SPR) measurements, affinity constants are known with antibody MabGO1:Nr1 kD=<10-3; Nr2 kD=3.10-7; Nr3 kD=4.10-6; and Nr4 kD=6.10-8.

[0057] Binding of the antibody to the peptides was detected using amethod that made use of a fluorescent product. The whole PP supportcontaining the peptide functionalized areas was incubated with theantibody (Mab GO1 5 ug/ml, incubation overnight). After washing, asubsequent incubation of a second anti-mouse antibody conjugated toalkaline phosphatase introduces, after binding of the Mab to thepeptide, the enzyme alkaline phosphatase at the peptide functionalizedsurface (spots). After washing, the bound enzyme caused fluorescentproduct signals at the peptide functionalized surfaces when a thin filmof a Vistra ECF substrate (Amersham Pharmacia Biotech) solution wasadded to the surface (excess substrate was removed). Fluorescent productsignals could be quantified on a Storm (Molecular Dynamics) in bluefluorescent mode.

[0058]FIG. 3 shows the Storm fluorescent signals of the binding of thepeptides Nr 1 (SEQ ID NO: 2), 2 (SEQ ID NO: 3), 3 (SEQ ID NO: 4) and 4(SEQ ID NO: 5) to Mab GO1 using five different gridding pins and fourdifferent peptide concentrations on 3 different grafts. FIGS. 4A-4D showthe maximal fluorescent signals of the spots on graft 6/12Ac. FIG. 5shows the maximal fluorescent signals of peptides Nr 1 (SEQ ID NO: 2), 2(SEQ ID NO: 3), 3 (SEQ ID NO: 4) and 4 (SEQ ID NO: 5) spotted with 0.2mg/ml on graft 6/12Ac, 9/30Ac and 12/50Ac.

Example 2

[0059] Glucose Oxidase. A polypropylene (PP) support (EVACAST 1070 N16;Vink Kunststoffen BV) was grafted with polyacrylic acid. The solidsupport was irradiated in the presence of 6% acrylic acid solution inwater containing CuSO4 using gamma radiation at a dose of 12kGy. Thegrafted solid support containing carboxylic acid groups wasfunctionalized with amino groups via coupling oft-butyloxycarbonylhexamethylenediamine (Boc-HMDA) usingdicyclohexylcarbodiimide (DCC) with N-hydroxybenztriazole (HOBt) andsubsequent cleavage of the Boc groups using trifluoracetic acid. Tointroduce a thiol reactive bromacetamide group on the support, the aminogroup functionalized support was treated with bromoacetic acid using DCCor DCC/HOBt.

[0060] Glucose oxidase containing thiol-groups (Glu-ox-SH) was able tocouple to the bromo functionalized surface. Thiol groups on Glucoseoxidase (Glu-ox; 1 mg/ml) were introduced in 0.16 M borate buffer (pH 8)using 2-iminothiolane (5 times molar excess 2-iminothiolane over Glu-ox;45 min at room temperature). Glu-ox-SH was spotted on the bromofunctionalized surface using gridding pins (Genomic Solutions) withdifferent diameters (1.5 mm, 0.8 mm, 0.6 mm, 0.4 mm and 0.25 mm).Concentration of Glu-ox-SH was 0.25 mg/ml. When aliquots of Glu-ox-SHsolutions (in phosphate buffered saline (PBS), 1 mM Titriplex=EDTA at pH7) were dispensed on the support using the gridding pins, the couplingof the bromo group of the surface to the thiol group of Glu-ox-SH wasachieved in a humid chamber (overnight reaction). Extensive washingremoved uncoupled Glu-ox-SH.

[0061] Binding of an antibody (Mab G01) to Glu-ox was detected using amethod that made use of a fluorescent product. The whole PP supportcontaining the Glu-ox functionalized areas was incubated with theantibody GO1 (5 ug/ml). After washing, a subsequent incubation of asecond anti mouse antibody conjugated to alkaline phosphataseintroduces, after binding of the Mab to Glu-ox, the enzyme alkalinephosphatase at the Glu-ox functionalized surface (spots). After washing,the bound enzyme caused fluorescent product signals at the peptidefunctionalized surfaces when Vistra ECF substrate (Amersham PharmaciaBiotech) (excess substrate was removed) was introduced. Fluorescentproduct signals could be quantified on a Storm (Molecular Dynamics) inblue fluorescent mode. FIG. 8 shows the Storm fluorescent signals of thebinding Glu-ox to Mab GO 1 using five different gridding pins and threedifferent grafts.

Example-3A

[0062] Head-to-tail matrix-scan. In a complete matrix-scan, theN-terminal sequence of, for instance, sequence (1-11) of a protein islinked as a building block with each overlapping peptide sequence of acomplete scan of the same protein as shown in FIG. 6A. Sequence (2-12)is linked with the same set of overlapping sequences and so on. The linkcan be formed, for instance, by reaction of a cysteine at the C-terminusof the second building block with a bromoacetamide modified N-terminusof the first building block. This means that every combination of, forinstance, undecapeptides from the protein sequence is synthesized on aseparate, known, position of the solid support.

Example-3B

[0063] (Type II): Tail-to-tail matrix-scan. This is the same scan as thecomplete matrix scan from Example 3A, however, in this scan, thecysteine of the second building block is located at its N-terminus andprovides a reversed or tail-to-tail orientation of both building blocksin the construct as shown in FIG. 6A. Examples 3A and 3B are illustratedin FIGS. 6B, 6C and 6D.

Example-4

[0064] Multi building block scan. In this example, a thiol function isintroduced on an amino-functionalized solid support. This can be made bya direct reaction of the amino groups with, for instance, iminothiolane,or by coupling of Fmoc-Cys(Trt)-OH, followed by Fmoc cleavage usingpiperidine, acetylation, and trityl deprotection using TFA/scavengermixtures. This thiol-functionalized solid support can be reacted with,for instance, a bromoacetamide-peptide, containing a protected cysteineresidue. After coupling of the first peptide, the cysteine can bedeprotected using, for instance, a TFA/scavenger mixture. The formedfree thiol group can be used to couple a second bromoacetamide-peptide,again containing a protected cysteine. This procedure can be repeated tomake multi-building block constructs. Several types of scans, asdescribed in the other examples, can be used in combination with thismulti building block scan. In FIG. 7A, an example is shown for a threemulti building block scan. A working example with two building blockscans is illustrated in FIGS. 7B, 7C and 7D.

REFERENCES

[0065] Frank R. Strategies and techniques in simultaneous solid phasesynthesis based on the segmentation of membrane type supports.Bioorganic and Medical Chemistry Letters, 1993 Vol. 3 Number 3 pages425-430.

[0066] Geysen H. M., Meloen R. H. and Barteling S. J. (1984). Use ofpeptide synthesis to probe viral antigens for epitopes to a resolutionof a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998-4002.

[0067] Slootstra J. W., Puijk W. C. Ligtvoet G. J., Langeveld J. P. M. &Meloen R. H. 1995. Structural aspects of antibody-antigen interactionrevealed through small random peptide libraries. Molecular Diversity 1,87-96.

[0068] Slootstra J W, Puijk W C, Ligtvoet G J, Kuperus D, Schaaper W MM, Meloen R H. 1997. Screening of a small set of random peptides: A newstrategy to identify peptides that mimic epitopes. J. Mol. Recog.10:219-224.

1 386 1 14 PRT artificial sequence artificial peptide building block 1Xaa Cys Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly 1 5 10 2 13 PRTartificial sequence artificial peptide Nr1 2 Gly Cys Ala Ser Leu Gln GlyMet Asp Thr Cys Gly Lys 1 5 10 3 12 PRT artificial sequence Artificialpeptide Nr2 3 Cys Ala Phe Lys Gln Gly Val Asp Thr Cys Gly Lys 1 5 10 413 PRT artificial sequence artificial peptide Nr3 4 Ala Pro Asp Pro PheGln Gly Val Asp Thr Cys Gly Lys 1 5 10 5 15 PRT artificial sequenceartificial peptide Nr4 5 Gly Cys Ala Pro Asp Pro Phe Gln Gly Val Asp ThrCys Gly Lys 1 5 10 15 6 12 PRT Homo sapiens 6 Ala Pro Asp Val Gln AspCys Pro Glu Cys Thr Leu 1 5 10 7 12 PRT Homo sapiens 7 Pro Asp Val GlnAsp Cys Pro Glu Cys Thr Leu Gln 1 5 10 8 12 PRT Homo sapiens 8 Asp ValGln Asp Cys Pro Glu Cys Thr Leu Gln Glu 1 5 10 9 12 PRT Homo sapiens 9Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn 1 5 10 10 12 PRT Homosapiens 10 Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro 1 5 10 11 12PRT Homo sapiens 11 Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe 1 510 12 12 PRT Homo sapiens 12 Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro PhePhe 1 5 10 13 12 PRT Homo sapiens 13 Pro Glu Cys Thr Leu Gln Glu Asn ProPhe Phe Ser 1 5 10 14 12 PRT Homo sapiens 14 Glu Cys Thr Leu Gln Glu AsnPro Phe Phe Ser Gln 1 5 10 15 12 PRT Homo sapiens 15 Cys Thr Leu Gln GluAsn Pro Phe Phe Ser Gln Pro 1 5 10 16 12 PRT Homo sapiens 16 Thr Leu GlnGlu Asn Pro Phe Phe Ser Gln Pro Gly 1 5 10 17 12 PRT Homo sapiens 17 LeuGln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala 1 5 10 18 12 PRT Homosapiens 18 Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 1 5 10 19 12PRT Homo sapiens 19 Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile 1 510 20 12 PRT Homo sapiens 20 Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro IleLeu 1 5 10 21 12 PRT Homo sapiens 21 Pro Phe Phe Ser Gln Pro Gly Ala ProIle Leu Gln 1 5 10 22 12 PRT Homo sapiens 22 Phe Phe Ser Gln Pro Gly AlaPro Ile Leu Gln Cys 1 5 10 23 12 PRT Homo sapiens 23 Phe Ser Gln Pro GlyAla Pro Ile Leu Gln Cys Met 1 5 10 24 12 PRT Homo sapiens 24 Ser Gln ProGly Ala Pro Ile Leu Gln Cys Met Gly 1 5 10 25 12 PRT Homo sapiens 25 GlnPro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys 1 5 10 26 12 PRT Homosapiens 26 Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys 1 5 10 27 12PRT Homo sapiens 27 Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe 1 510 28 12 PRT Homo sapiens 28 Ala Pro Ile Leu Gln Cys Met Gly Cys Cys PheSer 1 5 10 29 12 PRT Homo sapiens 29 Pro Ile Leu Gln Cys Met Gly Cys CysPhe Ser Arg 1 5 10 30 12 PRT Homo sapiens 30 Ile Leu Gln Cys Met Gly CysCys Phe Ser Arg Ala 1 5 10 31 12 PRT Homo sapiens 31 Leu Gln Cys Met GlyCys Cys Phe Ser Arg Ala Tyr 1 5 10 32 12 PRT Homo sapiens 32 Gln Cys MetGly Cys Cys Phe Ser Arg Ala Tyr Pro 1 5 10 33 12 PRT Homo sapiens 33 CysMet Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr 1 5 10 34 12 PRT Homosapiens 34 Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 1 5 10 35 12PRT Homo sapiens 35 Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu 1 510 36 12 PRT Homo sapiens 36 Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro LeuArg 1 5 10 37 12 PRT Homo sapiens 37 Cys Phe Ser Arg Ala Tyr Pro Thr ProLeu Arg Ser 1 5 10 38 12 PRT Homo sapiens 38 Phe Ser Arg Ala Tyr Pro ThrPro Leu Arg Ser Lys 1 5 10 39 12 PRT Homo sapiens 39 Ser Arg Ala Tyr ProThr Pro Leu Arg Ser Lys Lys 1 5 10 40 12 PRT Homo sapiens 40 Arg Ala TyrPro Thr Pro Leu Arg Ser Lys Lys Thr 1 5 10 41 12 PRT Homo sapiens 41 AlaTyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met 1 5 10 42 12 PRT Homosapiens 42 Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu 1 5 10 43 12PRT Homo sapiens 43 Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val 1 510 44 12 PRT Homo sapiens 44 Thr Pro Leu Arg Ser Lys Lys Thr Met Leu ValGln 1 5 10 45 12 PRT Homo sapiens 45 Pro Leu Arg Ser Lys Lys Thr Met LeuVal Gln Lys 1 5 10 46 12 PRT Homo sapiens 46 Leu Arg Ser Lys Lys Thr MetLeu Val Gln Lys Asn 1 5 10 47 12 PRT Homo sapiens 47 Arg Ser Lys Lys ThrMet Leu Val Gln Lys Asn Val 1 5 10 48 12 PRT Homo sapiens 48 Ser Lys LysThr Met Leu Val Gln Lys Asn Val Thr 1 5 10 49 12 PRT Homo sapiens 49 LysLys Thr Met Leu Val Gln Lys Asn Val Thr Ser 1 5 10 50 12 PRT Homosapiens 50 Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 1 5 10 51 12PRT Homo sapiens 51 Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser 1 510 52 12 PRT Homo sapiens 52 Met Leu Val Gln Lys Asn Val Thr Ser Glu SerThr 1 5 10 53 12 PRT Homo sapiens 53 Leu Val Gln Lys Asn Val Thr Ser GluSer Thr Cys 1 5 10 54 12 PRT Homo sapiens 54 Val Gln Lys Asn Val Thr SerGlu Ser Thr Cys Cys 1 5 10 55 12 PRT Homo sapiens 55 Gln Lys Asn Val ThrSer Glu Ser Thr Cys Cys Val 1 5 10 56 12 PRT Homo sapiens 56 Lys Asn ValThr Ser Glu Ser Thr Cys Cys Val Ala 1 5 10 57 12 PRT Homo sapiens 57 AsnVal Thr Ser Glu Ser Thr Cys Cys Val Ala Lys 1 5 10 58 12 PRT Homosapiens 58 Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser 1 5 10 59 12PRT Homo sapiens 59 Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr 1 510 60 12 PRT Homo sapiens 60 Ser Glu Ser Thr Cys Cys Val Ala Lys Ser TyrAsn 1 5 10 61 12 PRT Homo sapiens 61 Glu Ser Thr Cys Cys Val Ala Lys SerTyr Asn Arg 1 5 10 62 12 PRT Homo sapiens 62 Ser Thr Cys Cys Val Ala LysSer Tyr Asn Arg Val 1 5 10 63 12 PRT Homo sapiens 63 Thr Cys Cys Val AlaLys Ser Tyr Asn Arg Val Thr 1 5 10 64 12 PRT Homo sapiens 64 Cys Cys ValAla Lys Ser Tyr Asn Arg Val Thr Val 1 5 10 65 12 PRT Homo sapiens 65 CysVal Ala Lys Ser Tyr Asn Arg Val Thr Val Met 1 5 10 66 12 PRT Homosapiens 66 Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 1 5 10 67 12PRT Homo sapiens 67 Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly 1 510 68 12 PRT Homo sapiens 68 Lys Ser Tyr Asn Arg Val Thr Val Met Gly GlyPhe 1 5 10 69 12 PRT Homo sapiens 69 Ser Tyr Asn Arg Val Thr Val Met GlyGly Phe Lys 1 5 10 70 12 PRT Homo sapiens 70 Tyr Asn Arg Val Thr Val MetGly Gly Phe Lys Val 1 5 10 71 12 PRT Homo sapiens 71 Asn Arg Val Thr ValMet Gly Gly Phe Lys Val Glu 1 5 10 72 12 PRT Homo sapiens 72 Arg Val ThrVal Met Gly Gly Phe Lys Val Glu Asn 1 5 10 73 12 PRT Homo sapiens 73 ValThr Val Met Gly Gly Phe Lys Val Glu Asn His 1 5 10 74 12 PRT Homosapiens 74 Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr 1 5 10 75 12PRT Homo sapiens 75 Val Met Gly Gly Phe Lys Val Glu Asn His Thr Ala 1 510 76 12 PRT Homo sapiens 76 Met Gly Gly Phe Lys Val Glu Asn His Thr AlaCys 1 5 10 77 12 PRT Homo sapiens 77 Gly Gly Phe Lys Val Glu Asn His ThrAla Cys His 1 5 10 78 12 PRT Homo sapiens 78 Gly Phe Lys Val Glu Asn HisThr Ala Cys His Cys 1 5 10 79 12 PRT Homo sapiens 79 Phe Lys Val Glu AsnHis Thr Ala Cys His Cys Ser 1 5 10 80 12 PRT Homo sapiens 80 Lys Val GluAsn His Thr Ala Cys His Cys Ser Thr 1 5 10 81 12 PRT Homo sapiens 81 ValGlu Asn His Thr Ala Cys His Cys Ser Thr Cys 1 5 10 82 12 PRT Homosapiens 82 Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 1 5 10 83 12PRT Homo sapiens 83 Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr 1 510 84 12 PRT Homo sapiens 84 His Thr Ala Cys His Cys Ser Thr Cys Tyr TyrHis 1 5 10 85 12 PRT Homo sapiens 85 Thr Ala Cys His Cys Ser Thr Cys TyrTyr His Lys 1 5 10 86 12 PRT Homo sapiens 86 Ala Cys His Cys Ser Thr CysTyr Tyr His Lys Ser 1 5 10 87 12 PRT Homo sapiens 87 Asn Ser Cys Glu LeuThr Asn Ile Thr Ile Ala Ile 1 5 10 88 12 PRT Homo sapiens 88 Ser Cys GluLeu Thr Asn Ile Thr Ile Ala Ile Glu 1 5 10 89 12 PRT Homo sapiens 89 CysGlu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys 1 5 10 90 12 PRT Homosapiens 90 Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu 1 5 10 91 12PRT Homo sapiens 91 Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1 510 92 12 PRT Homo sapiens 92 Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu GluCys 1 5 10 93 12 PRT Homo sapiens 93 Asn Ile Thr Ile Ala Ile Glu Lys GluGlu Cys Arg 1 5 10 94 12 PRT Homo sapiens 94 Ile Thr Ile Ala Ile Glu LysGlu Glu Cys Arg Phe 1 5 10 95 12 PRT Homo sapiens 95 Thr Ile Ala Ile GluLys Glu Glu Cys Arg Phe Cys 1 5 10 96 12 PRT Homo sapiens 96 Ile Ala IleGlu Lys Glu Glu Cys Arg Phe Cys Ile 1 5 10 97 12 PRT Homo sapiens 97 AlaIle Glu Lys Glu Glu Cys Arg Phe Cys Ile Ser 1 5 10 98 12 PRT Homosapiens 98 Ala Ile Glu Lys Glu Glu Cys Arg Phe Cys Ile Ser 1 5 10 99 12PRT Homo sapiens 99 Glu Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn 1 510 100 12 PRT Homo sapiens 100 Lys Glu Glu Cys Arg Phe Cys Ile Ser IleAsn Thr 1 5 10 101 12 PRT Homo sapiens 101 Glu Glu Cys Arg Phe Cys IleSer Ile Asn Thr Thr 1 5 10 102 12 PRT Homo sapiens 102 Glu Cys Arg PheCys Ile Ser Ile Asn Thr Thr Trp 1 5 10 103 12 PRT Homo sapiens 103 CysArg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys 1 5 10 104 12 PRT Homosapiens 104 Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala 1 5 10 10512 PRT Homo sapiens 105 Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly1 5 10 106 12 PRT Homo sapiens 106 Cys Ile Ser Ile Asn Thr Thr Trp CysAla Gly Tyr 1 5 10 107 12 PRT Homo sapiens 107 Ile Ser Ile Asn Thr ThrTrp Cys Ala Gly Tyr Cys 1 5 10 108 12 PRT Homo sapiens 108 Ser Ile AsnThr Thr Trp Cys Ala Gly Tyr Cys Tyr 1 5 10 109 12 PRT Homo sapiens 109Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr 1 5 10 110 12 PRT Homosapiens 110 Asn Thr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr Arg 1 5 10 11112 PRT Homo sapiens 111 Thr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr Arg Asp1 5 10 112 12 PRT Homo sapiens 112 Thr Trp Cys Ala Gly Tyr Cys Tyr ThrArg Asp Leu 1 5 10 113 12 PRT Homo sapiens 113 Trp Cys Ala Gly Tyr CysTyr Thr Arg Asp Leu Val 1 5 10 114 12 PRT Homo sapiens 114 Cys Ala GlyTyr Cys Tyr Thr Arg Asp Leu Val Tyr 1 5 10 115 12 PRT Homo sapiens 115Ala Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys 1 5 10 116 12 PRT Homosapiens 116 Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp 1 5 10 11712 PRT Homo sapiens 117 Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro1 5 10 118 12 PRT Homo sapiens 118 Cys Tyr Thr Arg Asp Leu Val Tyr LysAsp Pro Ala 1 5 10 119 12 PRT Homo sapiens 119 Tyr Thr Arg Asp Leu ValTyr Lys Asp Pro Ala Arg 1 5 10 120 12 PRT Homo sapiens 120 Thr Arg AspLeu Val Tyr Lys Asp Pro Ala Arg Pro 1 5 10 121 12 PRT Homo sapiens 121Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys 1 5 10 122 12 PRT Homosapiens 122 Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile 1 5 10 12312 PRT Homo sapiens 123 Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln1 5 10 124 12 PRT Homo sapiens 124 Val Tyr Lys Asp Pro Ala Arg Pro LysIle Gln Lys 1 5 10 125 12 PRT Homo sapiens 125 Tyr Lys Asp Pro Ala ArgPro Lys Ile Gln Lys Thr 1 5 10 126 12 PRT Homo sapiens 126 Lys Asp ProAla Arg Pro Lys Ile Gln Lys Thr Cys 1 5 10 127 12 PRT Homo sapiens 127Asp Pro Ala Arg Pro Lys Ile Gln Lys Thr Cys Thr 1 5 10 128 12 PRT Homosapiens 128 Pro Ala Arg Pro Lys Ile Gln Lys Thr Cys Thr Phe 1 5 10 12912 PRT Homo sapiens 129 Ala Arg Pro Lys Ile Gln Lys Thr Cys Thr Phe Lys1 5 10 130 12 PRT Homo sapiens 130 Arg Pro Lys Ile Gln Lys Thr Cys ThrPhe Lys Glu 1 5 10 131 12 PRT Homo sapiens 131 Pro Lys Ile Gln Lys ThrCys Thr Phe Lys Glu Leu 1 5 10 132 12 PRT Homo sapiens 132 Lys Ile GlnLys Thr Cys Thr Phe Lys Glu Leu Val 1 5 10 133 12 PRT Homo sapiens 133Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr 1 5 10 134 12 PRT Homosapiens 134 Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu 1 5 10 13512 PRT Homo sapiens 135 Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr1 5 10 136 12 PRT Homo sapiens 136 Thr Cys Thr Phe Lys Glu Leu Val TyrGlu Thr Val 1 5 10 137 12 PRT Homo sapiens 137 Cys Thr Phe Lys Glu LeuVal Tyr Glu Thr Val Arg 1 5 10 138 12 PRT Homo sapiens 138 Thr Phe LysGlu Leu Val Tyr Glu Thr Val Arg Val 1 5 10 139 12 PRT Homo sapiens 139Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro 1 5 10 140 12 PRT Homosapiens 140 Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly 1 5 10 14112 PRT Homo sapiens 141 Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly Cys1 5 10 142 12 PRT Homo sapiens 142 Leu Val Tyr Glu Thr Val Arg Val ProGly Cys Ala 1 5 10 143 12 PRT Homo sapiens 143 Val Tyr Glu Thr Val ArgVal Pro Gly Cys Ala His 1 5 10 144 12 PRT Homo sapiens 144 Tyr Glu ThrVal Arg Val Pro Gly Cys Ala His His 1 5 10 145 12 PRT Homo sapiens 145Glu Thr Val Arg Val Pro Gly Cys Ala His His Ala 1 5 10 146 12 PRT Homosapiens 146 Thr Val Arg Val Pro Gly Cys Ala His His Ala Asp 1 5 10 14712 PRT Homo sapiens 147 Val Arg Val Pro Gly Cys Ala His His Ala Asp Ser1 5 10 148 12 PRT Homo sapiens 148 Arg Val Pro Gly Cys Ala His His AlaAsp Ser Leu 1 5 10 149 12 PRT Homo sapiens 149 Val Pro Gly Cys Ala HisHis Ala Asp Ser Leu Tyr 1 5 10 150 12 PRT Homo sapiens 150 Pro Gly CysAla His His Ala Asp Ser Leu Tyr Thr 1 5 10 151 12 PRT Homo sapiens 151Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr 1 5 10 152 12 PRT Homosapiens 152 Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro 1 5 10 15312 PRT Homo sapiens 153 Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val1 5 10 154 12 PRT Homo sapiens 154 His His Ala Asp Ser Leu Tyr Thr TyrPro Val Ala 1 5 10 155 12 PRT Homo sapiens 155 His Ala Asp Ser Leu TyrThr Tyr Pro Val Ala Thr 1 5 10 156 12 PRT Homo sapiens 156 Ala Asp SerLeu Tyr Thr Tyr Pro Val Ala Thr Gln 1 5 10 157 12 PRT Homo sapiens 157Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys 1 5 10 158 12 PRT Homosapiens 158 Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys His 1 5 10 15912 PRT Homo sapiens 159 Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys His Cys1 5 10 160 12 PRT Homo sapiens 160 Tyr Thr Tyr Pro Val Ala Thr Gln CysHis Cys Gly 1 5 10 161 12 PRT Homo sapiens 161 Thr Tyr Pro Val Ala ThrGln Cys His Cys Gly Lys 1 5 10 162 12 PRT Homo sapiens 162 Tyr Pro ValAla Thr Gln Cys His Cys Gly Lys Cys 1 5 10 163 12 PRT Homo sapiens 163Pro Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp 1 5 10 164 12 PRT Homosapiens 164 Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser 1 5 10 16512 PRT Homo sapiens 165 Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp1 5 10 166 12 PRT Homo sapiens 166 Thr Gln Cys His Cys Gly Lys Cys AspSer Asp Ser 1 5 10 167 12 PRT Homo sapiens 167 Gln Cys His Cys Gly LysCys Asp Ser Asp Ser Thr 1 5 10 168 12 PRT Homo sapiens 168 Cys His CysGly Lys Cys Asp Ser Asp Ser Thr Asp 1 5 10 169 12 PRT Homo sapiens 169His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys 1 5 10 170 12 PRT Homosapiens 170 Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr 1 5 10 17112 PRT Homo sapiens 171 Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val1 5 10 172 12 PRT Homo sapiens 172 Lys Cys Asp Ser Asp Ser Thr Asp CysThr Val Arg 1 5 10 173 12 PRT Homo sapiens 173 Cys Asp Ser Asp Ser ThrAsp Cys Thr Val Arg Gly 1 5 10 174 12 PRT Homo sapiens 174 Asp Ser AspSer Thr Asp Cys Thr Val Arg Gly Leu 1 5 10 175 12 PRT Homo sapiens 175Ser Asp Ser Thr Asp Cys Thr Val Arg Gly Leu Gly 1 5 10 176 12 PRT Homosapiens 176 Asp Ser Thr Asp Cys Thr Val Arg Gly Leu Gly Pro 1 5 10 17712 PRT Homo sapiens 177 Ser Thr Asp Cys Thr Val Arg Gly Leu Gly Pro Ser1 5 10 178 12 PRT Homo sapiens 178 Thr Asp Cys Thr Val Arg Gly Leu GlyPro Ser Tyr 1 5 10 179 12 PRT Homo sapiens 179 Asp Cys Thr Val Arg GlyLeu Gly Pro Ser Tyr Cys 1 5 10 180 12 PRT Homo sapiens 180 Cys Thr ValArg Gly Leu Gly Pro Ser Tyr Cys Ser 1 5 10 181 12 PRT Homo sapiens 181Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe 1 5 10 182 12 PRT Homosapiens 182 Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly 1 5 10 18312 PRT Homo sapiens 183 Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu1 5 10 184 12 PRT Homo sapiens 184 Gly Leu Gly Pro Ser Tyr Cys Ser PheGly Glu Met 1 5 10 185 12 PRT Homo sapiens 185 Leu Gly Pro Ser Tyr CysSer Phe Gly Glu Met Lys 1 5 10 186 12 PRT Homo sapiens 186 Gly Pro SerTyr Cys Ser Phe Gly Glu Met Lys Glu 1 5 10 187 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 187 Ala ProAsp Val Gln Asp Cys Pro Glu Cys Thr Cys 1 5 10 188 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 188 Cys ProAsp Val Gln Asp Cys Pro Glu Cys Thr Leu 1 5 10 189 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 189 Asp ValGln Asp Cys Pro Glu Cys Thr Leu Gln Cys 1 5 10 190 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 190 Cys ValGln Asp Cys Pro Glu Cys Thr Leu Gln Glu 1 5 10 191 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 191 Gln AspCys Pro Glu Cys Thr Leu Gln Glu Asn Cys 1 5 10 192 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 192 Cys AspCys Pro Glu Cys Thr Leu Gln Glu Asn Pro 1 5 10 193 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 193 Cys ProGlu Cys Thr Leu Gln Glu Asn Pro Phe Cys 1 5 10 194 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 194 Glu CysThr Leu Gln Glu Asn Pro Phe Phe Ser Cys 1 5 10 195 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 195 Cys CysThr Leu Gln Glu Asn Pro Phe Phe Ser Gln 1 5 10 196 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 196 Thr LeuGln Glu Asn Pro Phe Phe Ser Gln Pro Cys 1 5 10 197 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 197 Cys LeuGln Glu Asn Pro Phe Phe Ser Gln Pro Gly 1 5 10 198 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 198 Gln GluAsn Pro Phe Phe Ser Gln Pro Gly Ala Cys 1 5 10 199 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 199 Cys GluAsn Pro Phe Phe Ser Gln Pro Gly Ala Pro 1 5 10 200 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 200 Asn ProPhe Phe Ser Gln Pro Gly Ala Pro Ile Cys 1 5 10 201 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 201 Cys ProPhe Phe Ser Gln Pro Gly Ala Pro Ile Leu 1 5 10 202 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 202 Cys PheSer Gln Pro Gly Ala Pro Ile Leu Gln Cys 1 5 10 203 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 203 Ser GlnPro Gly Ala Pro Ile Leu Gln Cys Met Cys 1 5 10 204 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 204 Cys GlnPro Gly Ala Pro Ile Leu Gln Cys Met Gly 1 5 10 205 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 205 Cys GlyAla Pro Ile Leu Gln Cys Met Gly Cys Cys 1 5 10 206 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 206 Ala ProIle Leu Gln Cys Met Gly Cys Cys Phe Cys 1 5 10 207 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 207 Cys ProIle Leu Gln Cys Met Gly Cys Cys Phe Ser 1 5 10 208 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 208 Ile LeuGln Cys Met Gly Cys Cys Phe Ser Arg Cys 1 5 10 209 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 209 Cys LeuGln Cys Met Gly Cys Cys Phe Ser Arg Ala 1 5 10 210 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 210 Gln CysMet Gly Cys Cys Phe Ser Arg Ala Tyr Cys 1 5 10 211 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 211 Cys CysMet Gly Cys Cys Phe Ser Arg Ala Tyr Pro 1 5 10 212 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 212 Met GlyCys Cys Phe Ser Arg Ala Tyr Pro Thr Cys 1 5 10 213 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 213 Cys GlyCys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 1 5 10 214 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 214 Cys CysPhe Ser Arg Ala Tyr Pro Thr Pro Leu Cys 1 5 10 215 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 215 Phe SerArg Ala Tyr Pro Thr Pro Leu Arg Ser Cys 1 5 10 216 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 216 Cys SerArg Ala Tyr Pro Thr Pro Leu Arg Ser Lys 1 5 10 217 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 217 Arg AlaTyr Pro Thr Pro Leu Arg Ser Lys Lys Cys 1 5 10 218 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 218 Cys AlaTyr Pro Thr Pro Leu Arg Ser Lys Lys Thr 1 5 10 219 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 219 Tyr ProThr Pro Leu Arg Ser Lys Lys Thr Met Cys 1 5 10 220 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 220 Cys ProThr Pro Leu Arg Ser Lys Lys Thr Met Leu 1 5 10 221 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 221 Thr ProLeu Arg Ser Lys Lys Thr Met Leu Val Cys 1 5 10 222 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 222 Cys ProLeu Arg Ser Lys Lys Thr Met Leu Val Gln 1 5 10 223 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 223 Leu ArgSer Lys Lys Thr Met Leu Val Gln Lys Cys 1 5 10 224 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 224 Cys ArgSer Lys Lys Thr Met Leu Val Gln Lys Asn 1 5 10 225 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 225 Ser LysLys Thr Met Leu Val Gln Lys Asn Val Cys 1 5 10 226 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 226 Cys LysLys Thr Met Leu Val Gln Lys Asn Val Thr 1 5 10 227 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 227 Lys ThrMet Leu Val Gln Lys Asn Val Thr Ser Cys 1 5 10 228 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 228 Cys ThrMet Leu Val Gln Lys Asn Val Thr Ser Glu 1 5 10 229 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 229 Met LeuVal Gln Lys Asn Val Thr Ser Glu Ser Cys 1 5 10 230 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 230 Cys LeuVal Gln Lys Asn Val Thr Ser Glu Ser Thr 1 5 10 231 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 231 Cys GlnLys Asn Val Thr Ser Glu Ser Thr Cys Cys 1 5 10 232 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 232 Lys AsnVal Thr Ser Glu Ser Thr Cys Cys Val Cys 1 5 10 233 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 233 Cys AsnVal Thr Ser Glu Ser Thr Cys Cys Val Ala 1 5 10 234 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 234 Val ThrSer Glu Ser Thr Cys Cys Val Ala Lys Cys 1 5 10 235 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 235 Cys ThrSer Glu Ser Thr Cys Cys Val Ala Lys Ser 1 5 10 236 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 236 Ser GluSer Thr Cys Cys Val Ala Lys Ser Tyr Cys 1 5 10 237 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 237 Cys GluSer Thr Cys Cys Val Ala Lys Ser Tyr Asn 1 5 10 238 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 238 Ser ThrCys Cys Val Ala Lys Ser Tyr Asn Arg Cys 1 5 10 239 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 239 Cys ThrCys Cys Val Ala Lys Ser Tyr Asn Arg Val 1 5 10 240 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 240 Cys CysVal Ala Lys Ser Tyr Asn Arg Val Thr Cys 1 5 10 241 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 241 Val AlaLys Ser Tyr Asn Arg Val Thr Val Met Cys 1 5 10 242 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 242 Cys AlaLys Ser Tyr Asn Arg Val Thr Val Met Gly 1 5 10 243 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 243 Lys SerTyr Asn Arg Val Thr Val Met Gly Gly Cys 1 5 10 244 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 244 Cys SerTyr Asn Arg Val Thr Val Met Gly Gly Phe 1 5 10 245 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 245 Tyr AsnArg Val Thr Val Met Gly Gly Phe Lys Cys 1 5 10 246 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 246 Cys AsnArg Val Thr Val Met Gly Gly Phe Lys Val 1 5 10 247 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 247 Arg ValThr Val Met Gly Gly Phe Lys Val Glu Cys 1 5 10 248 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 248 Cys ValThr Val Met Gly Gly Phe Lys Val Glu Asn 1 5 10 249 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 249 Thr ValMet Gly Gly Phe Lys Val Glu Asn His Cys 1 5 10 250 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 250 Cys ValMet Gly Gly Phe Lys Val Glu Asn His Thr 1 5 10 251 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 251 Cys GlyGly Phe Lys Val Glu Asn His Thr Ala Cys 1 5 10 252 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 252 Cys PheLys Val Glu Asn His Thr Ala Cys His Cys 1 5 10 253 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 253 Lys ValGlu Asn His Thr Ala Cys His Cys Ser Cys 1 5 10 254 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 254 Cys ValGlu Asn His Thr Ala Cys His Cys Ser Thr 1 5 10 255 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 255 Glu AsnHis Thr Ala Cys His Cys Ser Thr Cys Cys 1 5 10 256 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 256 Cys AsnHis Thr Ala Cys His Cys Ser Thr Cys Tyr 1 5 10 257 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 257 His ThrAla Cys His Cys Ser Thr Cys Tyr Tyr Cys 1 5 10 258 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 258 Cys ThrAla Cys His Cys Ser Thr Cys Tyr Tyr His 1 5 10 259 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 259 Ala CysHis Cys Ser Thr Cys Tyr Tyr His Lys Cys 1 5 10 260 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 260 Cys CysHis Cys Ser Thr Cys Tyr Tyr His Lys Ser 1 5 10 261 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 261 Asn SerCys Glu Leu Thr Asn Ile Thr Ile Ala Cys 1 5 10 262 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 262 Cys SerCys Glu Leu Thr Asn Ile Thr Ile Ala Ile 1 5 10 263 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 263 Cys GluLeu Thr Asn Ile Thr Ile Ala Ile Glu Cys 1 5 10 264 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 264 Leu ThrAsn Ile Thr Ile Ala Ile Glu Lys Glu Cys 1 5 10 265 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 265 Cys ThrAsn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1 5 10 266 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 266 Asn IleThr Ile Ala Ile Glu Lys Glu Glu Cys Cys 1 5 10 267 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 267 Cys IleThr Ile Ala Ile Glu Lys Glu Glu Cys Arg 1 5 10 268 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 268 Cys IleAla Ile Glu Lys Glu Glu Cys Arg Phe Cys 1 5 10 269 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 269 Ala IleGlu Lys Glu Glu Cys Arg Phe Cys Ile Cys 1 5 10 270 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 270 Cys IleGlu Lys Glu Glu Cys Arg Phe Cys Ile Ser 1 5 10 271 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 271 Glu LysGlu Glu Cys Arg Phe Cys Ile Ser Ile Cys 1 5 10 272 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 272 Cys LysGlu Glu Cys Arg Phe Cys Ile Ser Ile Asn 1 5 10 273 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 273 Glu GluCys Arg Phe Cys Ile Ser Ile Asn Thr Cys 1 5 10 274 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 274 Cys GluCys Arg Phe Cys Ile Ser Ile Asn Thr Thr 1 5 10 275 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 275 Phe CysIle Ser Ile Asn Thr Thr Trp Cys Ala Cys 1 5 10 276 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 276 Cys CysIle Ser Ile Asn Thr Thr Trp Cys Ala Gly 1 5 10 277 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 277 Cys SerIle Asn Thr Thr Trp Cys Ala Gly Tyr Cys 1 5 10 278 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 278 Ile AsnThr Thr Trp Cys Ala Gly Tyr Cys Tyr Cys 1 5 10 279 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 279 Cys AsnThr Thr Trp Cys Ala Gly Tyr Cys Tyr Thr 1 5 10 280 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 280 Thr ThrTrp Cys Ala Gly Tyr Cys Tyr Thr Arg Cys 1 5 10 281 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 281 Cys ThrTrp Cys Ala Gly Tyr Cys Tyr Thr Arg Asp 1 5 10 282 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 282 Trp CysAla Gly Tyr Cys Tyr Thr Arg Asp Leu Cys 1 5 10 283 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 283 Cys CysAla Gly Tyr Cys Tyr Thr Arg Asp Leu Val 1 5 10 284 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 284 Ala GlyTyr Cys Tyr Thr Arg Asp Leu Val Tyr Cys 1 5 10 285 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 285 Cys GlyTyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys 1 5 10 286 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 286 Tyr CysTyr Thr Arg Asp Leu Val Tyr Lys Asp Cys 1 5 10 287 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 287 Cys CysTyr Thr Arg Asp Leu Val Tyr Lys Asp Pro 1 5 10 288 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 288 Tyr ThrArg Asp Leu Val Tyr Lys Asp Pro Ala Cys 1 5 10 289 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 289 Cys ThrArg Asp Leu Val Tyr Lys Asp Pro Ala Arg 1 5 10 290 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 290 Arg AspLeu Val Tyr Lys Asp Pro Ala Arg Pro Cys 1 5 10 291 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 291 Cys AspLeu Val Tyr Lys Asp Pro Ala Arg Pro Lys 1 5 10 292 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 292 Leu ValTyr Lys Asp Pro Ala Arg Pro Lys Ile Cys 1 5 10 293 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 293 Cys ValTyr Lys Asp Pro Ala Arg Pro Lys Ile Gln 1 5 10 294 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 294 Tyr LysAsp Pro Ala Arg Pro Lys Ile Gln Lys Cys 1 5 10 295 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 295 Cys LysAsp Pro Ala Arg Pro Lys Ile Gln Lys Thr 1 5 10 296 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 296 Asp ProAla Arg Pro Lys Ile Gln Lys Thr Cys Cys 1 5 10 297 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 297 Cys ProAla Arg Pro Lys Ile Gln Lys Thr Cys Thr 1 5 10 298 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 298 Ala ArgPro Lys Ile Gln Lys Thr Cys Thr Phe Cys 1 5 10 299 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 299 Cys ArgPro Lys Ile Gln Lys Thr Cys Thr Phe Lys 1 5 10 300 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 300 Pro LysIle Gln Lys Thr Cys Thr Phe Lys Glu Cys 1 5 10 301 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 301 Cys LysIle Gln Lys Thr Cys Thr Phe Lys Glu Leu 1 5 10 302 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 302 Ile GlnLys Thr Cys Thr Phe Lys Glu Leu Val Cys 1 5 10 303 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 303 Cys GlnLys Thr Cys Thr Phe Lys Glu Leu Val Tyr 1 5 10 304 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 304 Lys ThrCys Thr Phe Lys Glu Leu Val Tyr Glu Cys 1 5 10 305 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 305 Cys ThrCys Thr Phe Lys Glu Leu Val Tyr Glu Thr 1 5 10 306 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 306 Cys ThrPhe Lys Glu Leu Val Tyr Glu Thr Val Cys 1 5 10 307 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 307 Phe LysGlu Leu Val Tyr Glu Thr Val Arg Val Cys 1 5 10 308 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 308 Cys LysGlu Leu Val Tyr Glu Thr Val Arg Val Pro 1 5 10 309 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 309 Cys LeuVal Tyr Glu Thr Val Arg Val Pro Gly Cys 1 5 10 310 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 310 Val TyrGlu Thr Val Arg Val Pro Gly Cys Ala Cys 1 5 10 311 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 311 Cys TyrGlu Thr Val Arg Val Pro Gly Cys Ala His 1 5 10 312 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 312 Glu ThrVal Arg Val Pro Gly Cys Ala His His Cys 1 5 10 313 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 313 Cys ThrVal Arg Val Pro Gly Cys Ala His His Ala 1 5 10 314 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 314 Val ArgVal Pro Gly Cys Ala His His Ala Asp Cys 1 5 10 315 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 315 Cys ArgVal Pro Gly Cys Ala His His Ala Asp Ser 1 5 10 316 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 316 Val ProGly Cys Ala His His Ala Asp Ser Leu Cys 1 5 10 317 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 317 Cys ProGly Cys Ala His His Ala Asp Ser Leu Tyr 1 5 10 318 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 318 Gly CysAla His His Ala Asp Ser Leu Tyr Thr Cys 1 5 10 319 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 319 Cys CysAla His His Ala Asp Ser Leu Tyr Thr Tyr 1 5 10 320 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 320 Ala HisHis Ala Asp Ser Leu Tyr Thr Tyr Pro Cys 1 5 10 321 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 321 Cys HisHis Ala Asp Ser Leu Tyr Thr Tyr Pro Val 1 5 10 322 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 322 His AlaAsp Ser Leu Tyr Thr Tyr Pro Val Ala Cys 1 5 10 323 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 323 Cys AlaAsp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 1 5 10 324 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 324 Cys SerLeu Tyr Thr Tyr Pro Val Ala Thr Gln Cys 1 5 10 325 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 325 Cys TyrThr Tyr Pro Val Ala Thr Gln Cys His Cys 1 5 10 326 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 326 Thr TyrPro Val Ala Thr Gln Cys His Cys Gly Cys 1 5 10 327 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 327 Cys TyrPro Val Ala Thr Gln Cys His Cys Gly Lys 1 5 10 328 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 328 Pro ValAla Thr Gln Cys His Cys Gly Lys Cys Cys 1 5 10 329 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 329 Cys ValAla Thr Gln Cys His Cys Gly Lys Cys Asp 1 5 10 330 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 330 Ala ThrGln Cys His Cys Gly Lys Cys Asp Ser Cys 1 5 10 331 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 331 Cys ThrGln Cys His Cys Gly Lys Cys Asp Ser Asp 1 5 10 332 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 332 Gln CysHis Cys Gly Lys Cys Asp Ser Asp Ser Cys 1 5 10 333 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 333 Cys CysHis Cys Gly Lys Cys Asp Ser Asp Ser Thr 1 5 10 334 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 334 Cys CysGly Lys Cys Asp Ser Asp Ser Thr Asp Cys 1 5 10 335 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 335 Gly LysCys Asp Ser Asp Ser Thr Asp Cys Thr Cys 1 5 10 336 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 336 Cys LysCys Asp Ser Asp Ser Thr Asp Cys Thr Val 1 5 10 337 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 337 Cys AspSer Asp Ser Thr Asp Cys Thr Val Arg Cys 1 5 10 338 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 338 Ser AspSer Thr Asp Cys Thr Val Arg Gly Leu Cys 1 5 10 339 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 339 Cys AspSer Thr Asp Cys Thr Val Arg Gly Leu Gly 1 5 10 340 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 340 Ser ThrAsp Cys Thr Val Arg Gly Leu Gly Pro Cys 1 5 10 341 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 341 Cys ThrAsp Cys Thr Val Arg Gly Leu Gly Pro Ser 1 5 10 342 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 342 Cys CysThr Val Arg Gly Leu Gly Pro Ser Tyr Cys 1 5 10 343 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 343 Thr ValArg Gly Leu Gly Pro Ser Tyr Cys Ser Cys 1 5 10 344 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 344 Cys ValArg Gly Leu Gly Pro Ser Tyr Cys Ser Phe 1 5 10 345 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 345 Arg GlyLeu Gly Pro Ser Tyr Cys Ser Phe Gly Cys 1 5 10 346 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 346 Cys GlyLeu Gly Pro Ser Tyr Cys Ser Phe Gly Glu 1 5 10 347 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 347 Leu GlyPro Ser Tyr Cys Ser Phe Gly Glu Met Cys 1 5 10 348 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 348 Cys GlyPro Ser Tyr Cys Ser Phe Gly Glu Met Lys 1 5 10 349 12 PRT ArtificialFragment of hFSH with Cys attached to the C or N terminal 349 Pro SerTyr Cys Ser Phe Gly Glu Met Lys Glu Cys 1 5 10 350 25 PRT ArtificialConcatonated sequences of hFSH 350 Val Tyr Glu Thr Val Arg Val Pro GlyCys Ala Cys Xaa Ala Asp Ser 1 5 10 15 Leu Tyr Thr Tyr Pro Val Ala ThrGln 20 25 351 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 351 Lys Thr Ala Thr Phe Lys Glu Leu Val Tyr Glu ThrCys 1 5 10 352 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 352 Cys Thr Ala Thr Phe Lys Glu Leu Val Tyr Glu ThrVal 1 5 10 353 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 353 Ala Thr Phe Lys Glu Leu Val Tyr Glu Thr Val ArgCys 1 5 10 354 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 354 Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val ArgVal 1 5 10 355 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 355 Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val ProCys 1 5 10 356 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 356 Cys Lys Glu Leu Val Tyr Glu Thr Val Arg Val ProGly 1 5 10 357 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 357 Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly AlaCys 1 5 10 358 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 358 Cys Leu Val Tyr Glu Thr Val Arg Val Pro Gly AlaAla 1 5 10 359 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 359 Val Tyr Glu Thr Val Arg Val Pro Gly Ala Ala HisCys 1 5 10 360 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 360 Cys Tyr Glu Thr Val Arg Val Pro Gly Ala Ala HisHis 1 5 10 361 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 361 Glu Thr Val Arg Val Pro Gly Ala Ala His His AlaCys 1 5 10 362 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 362 Cys Thr Val Arg Val Pro Gly Ala Ala His His AlaAsp 1 5 10 363 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 363 Val Arg Val Pro Gly Ala Ala His His Ala Asp SerCys 1 5 10 364 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 364 Cys Arg Val Pro Gly Ala Ala His His Ala Asp SerLeu 1 5 10 365 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 365 Val Pro Gly Ala Ala His His Ala Asp Ser Leu TyrCys 1 5 10 366 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 366 Cys Pro Gly Ala Ala His His Ala Asp Ser Leu TyrThr 1 5 10 367 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 367 Gly Ala Ala His His Ala Asp Ser Leu Tyr Thr TyrCys 1 5 10 368 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 368 Cys Ala Ala His His Ala Asp Ser Leu Tyr Thr TyrPro 1 5 10 369 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 369 Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro ValCys 1 5 10 370 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 370 Cys His His Ala Asp Ser Leu Tyr Thr Tyr Pro ValAla 1 5 10 371 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 371 His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala ThrCys 1 5 10 372 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 372 Cys Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala ThrGln 1 5 10 373 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 373 Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln AlaCys 1 5 10 374 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 374 Cys Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln AlaHis 1 5 10 375 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 375 Leu Tyr Thr Tyr Pro Val Ala Thr Gln Ala His AlaCys 1 5 10 376 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 376 Cys Tyr Thr Tyr Pro Val Ala Thr Gln Ala His AlaGly 1 5 10 377 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 377 Thr Tyr Pro Val Ala Thr Gln Ala His Ala Gly LysCys 1 5 10 378 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 378 Cys Tyr Pro Val Ala Thr Gln Ala His Ala Gly LysAla 1 5 10 379 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 379 Pro Val Ala Thr Gln Ala His Ala Gly Lys Ala AspCys 1 5 10 380 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 380 Cys Val Ala Thr Gln Ala His Ala Gly Lys Ala AspSer 1 5 10 381 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 381 Ala Thr Gln Ala His Ala Gly Lys Ala Asp Ser AspCys 1 5 10 382 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 382 Cys Thr Gln Ala His Ala Gly Lys Ala Asp Ser AspSer 1 5 10 383 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 383 Gln Ala His Ala Gly Lys Ala Asp Ser Asp Ser ThrCys 1 5 10 384 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 384 Cys Ala His Ala Gly Lys Ala Asp Ser Asp Ser ThrAsp 1 5 10 385 13 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 385 Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr GlnCys 1 5 10 386 10 PRT Artificial Fragment of hFSH with Cys attached tothe C or N terminal 386 Val Tyr Glu Thr Val Arg Val Pro Gly Cys 1 5 10

What is claimed is:
 1. A micro-array support for determining binding ofa first member molecule within a library of spots of tentative firstmember binding molecules with a second member binding molecule, themicro-array support comprising: a support surface having surface areas;and surface patches interspersed within the surface areas; wherein thesurface areas are materially distinct from the surface patches.
 2. Themicro-array support of claim 1, wherein the surface patches arehydrophilic and the surface areas are hydrophobic.
 3. The micro-arraysupport of claim 1 or 2, wherein: the surface areas consist essentiallyof hydrophobic polypropylene; and the surface patches consistessentially of polypropylene and a hydrophilic material.
 4. Themicro-array support of claim 3, wherein the hydrophilic materialcomprises polyacrylic acid.
 5. The micro-array support of any one ofclaims 1 to 4, further comprising a library of tentative first memberbinding molecules arranged in spatially addressable spots on the supportsurface.
 6. The micro-array support of any one of claims 1 to 5, furthercomprising a spot density of at least 25 spots per square centimeter. 7.A process for determining binding of a first member molecule within alibrary of tentative first member binding molecules with a second memberbinding molecule, the process comprising: providing the micro-arraysupport of any one of claims 1 to 6 with spots comprising the tentativefirst member binding molecules; providing a second member bindingmolecule; and detecting binding of the first member molecule with thesecond member binding molecule.
 8. The process according to claim 7,wherein the binding is detected with an optically detectable marker. 9.The process according to claim 8, wherein the optically detectablemarker comprises a fluorophore.
 10. The process according to claim 9,wherein the binding is detected with an enzyme-linked-assay.
 11. Theprocess according to claim 10, wherein the enzyme-linked-assay comprisesthe production of a fluorescent substrate.
 12. The process according toclaim 11, wherein an enzyme of the enzyme-linked-assay comprisesalkaline phosphatase.
 13. A process for determining binding of a firstmember molecule within a library of tentative first member bindingmolecules with a second member binding molecule, the process comprising:providing a micro-array support having a library of spots of thetentative first member binding molecules; wherein the library of spotsincludes a density of at least twenty-five spots per square centimeteron a support surface of the micro-array support; placing the secondmember binding molecule in contact with the library of the spots; anddetecting binding between the first member molecule and the secondbinding molecule with an enzyme-linked assay.
 14. The process accordingto claim 13, wherein the enzyme-linked-assay comprises the production ofa fluorescent substrate.
 15. The process according to claim 14, whereinan enzyme of the enzyme-linked assay comprises alkaline phosphatase. 16.A process for determining binding of a first member molecule within alibrary of tentative first member binding molecules with a second memberbinding molecule comprising: providing a micro-array support having alibrary of spots of the tentative first member binding molecules;placing the second member binding molecule in contact with the libraryof spots; detecting binding between the first member molecule and thesecond member biding molecule with an enzyme linked assay; and providingfor limited diffusion of an optically detectable marker molecule. 17.The process according to claim 16, wherein providing for limiteddiffusion comprises providing a support surface of the micro-arraysupport with surface patches interspersed within surface areas of thesupport surface, wherein the surface areas are materially distinct fromthe surface patches.
 18. The process according to claim 17, wherein thesurface patches are hydrophilic and the surface areas are hydrophobic.19. The process according to claim 17 or 18, wherein the surface areasconsist essentially of hydrophobic polypropylene and the surface patchesconsist essentially of polypropylene and a hydrophilic material.
 20. Theprocess according to claim 19, wherein the hydrophilic materialcomprises polyacrylic acid.
 21. The process according to any one ofclaims 13 to 20, wherein each spot of the library of spots are spatiallyaddressable.
 22. A synthetic molecule comprising a binding site, thesynthetic molecule identified or produced by the process according toany one of claims 7 to
 21. 23. A binding molecule comprising a bindingsite, the binding molecule identified or produced by the processaccording to any one of claims 7 to
 21. 24. A process for identifying orobtaining a synthetic molecule having a binding site, the processcomprising: providing the micro-array support of any one of claims 1 to6 with spots comprising the tentative first member binding molecules;providing a second member binding molecule; and detecting binding of thefirst member molecule with the second member binding molecule.
 25. Aprocess for identifying or obtaining a binding molecule capable ofbinding to a binding site, the process comprising: providing themicro-array support of any one of claims 1 to 6 with spots comprisingthe tentative first member binding molecules; providing a second memberbinding molecule; and detecting binding of the first member moleculewith the second member binding molecule.
 26. A process for interferingwith or affecting binding to a binding molecule or a synthetic molecule,the process comprising: providing the synthetic molecule of claim 22,the binding molecule of claim 23 or the synthetic molecule identified orproduced by the process of claim 24; and altering the synthetic moleculeof claim 22, the binding molecule of claim 23 or the synthetic moleculeidentified or produced by the process of claim 24, such that binding tothe synthetic molecule of claim 22, the binding molecule of claim 23 orthe synthetic molecule identified or produced by the process of claim 24is interfered with or affected.